Shewanella colwelliana are periphytic, gram-negative marine bacteria originally isolated from the walls of oyster spat tanks. The bacterium, originally designated LST (Lewis Spat Tank isolate), was temporarily assigned to the genus Alteromonas (R. M. Weiner, V. E. Coyne, P. Brayton, P. West, and S. F. Raiken (1988) Int. J. System. Bact. 38:240-244), and has now been reclassified to the recently established genus Shewanella (V. E. Coyne, C. J. Pillidge, D. D. Sledjeski, H. Hori, B. A. Ortiz-Conde, D. G. Muir, R. M. Weiner, and R. R. Colwell (1989) System. Appl. Microbiol. 12:275-279). These bacteria synthesize melanin, a brown-black polymeric pigment, and melanin metabolite precursors associated with an exopolysaccharide capable of inducing settlement and metamorphosis of oyster larvae, especially Crassostrea virginica larvae. Melanin synthesis occurs during late logarithmic and early stationary stages of growth.
Melanin is a general class of dark pigments found in bacteria, fungi, and higher organisms. This broad class of pigment is composed of complex polymers synthesized from phenolic or polyphenolic compounds (A. A. Bell and M. H. Wheeler (1986) Ann. Rev. Phytopathol. 24:411-451). The polymer is highly recalcitrant to degradation, and is observed frequently in the environment (i.e., humic soil deposits). Melanin is thought to be a "stable" free radical because it contains semiquinone free radicals that are stabilized by quinone and hydroxyquinone constituents of the polymer (A. A. Bell and M. H. Wheeler (1986) An. Rev. Phytopathol. 24:411-451). In addition, due to its complex composition melanin is capable of acting as either an oxidant or a reductant (M. S. Blois (1971) p. 125-139 in T. Kawamura, T. B. Fitzpatrick, and M. Seiji (eds), Biology of Normal and Abnormal Melanocytes Univ. Tokyo Press, Tokyo).
The classic Mason-Raper biosynthetic pathway for the synthesis of DOPA melanin was proposed in the late 1920's for one class of melanin (H. S. Raper (1928) Physiol. Rev. 8:245-282). In this pathway tyrosine is converted to DOPA via ortho-hydroxylation, and DOPA is then oxidized at both ring hydroxyl groups to form dopaquinone.
Melanogenesis has been documented for a number of bacterial species such as Streptomyces, Bacillus, Rhizobium, Legionella, and Vibrio; from fungi such as Neurospora and mushrooms; from amphibians such as Xenopus; and from mammals such as mice and humans. By far the best studied enzymes known to mediate melanogenesis are the tyrosinases (also known as catechol oxidase, phenolase, and polyphenol oxidase). Some of the genes encoding these tyrosinases have been cloned and sequenced but until the present invention no tyrosinase gene from a gram-negative bacterium or marine microorganism has been cloned or even isolated. In particular, cloned and sequenced tyrosinase genes have been reported from Streptomyces antibioticus, from Streptomyces glaucescens, from mouse pigment cells, and from human melanocytes [Bernan, V. et al. (1985) Gene 37:101-110; Hintermann, G. et al. (1985) Mol. Gen. Genet. 200:422-432; Shibahara, S. et al. (1986) Nucleic Acids Res. 14: 2413-2427; Kwon, B. S. et al. (1987) Proc. Natl. Acad. Sci. USA 84:7473-7477]. Additionally, the protein sequence of the Neurospora crassa tyrosinase has been determined [Lerch, K. (1982) J. Biol. Chem. 257:6414-6419].
Almost all genetic investigations in animals have focused on the mouse and human genes. A number of genetic loci have been defined in mice, where the A.sup.Y (lethal yellow) and e (extension) loci affect whether the melanin is black or yellow (sulfhydryl phaomelanin). Other loci, the b (brown), p (pink), and c (albino) appear to modulate the levels of tyrosinase activity in melanocytes (M. Jimenez, K. Tsukamoto, and V. J. Hearing (1991) J. Biol. Chem. 266:1147-1156). A gene encoding tyrosinase, corresponding to the b locus, was isolated from a cDNA library generated from B16 mouse melanoma cells (S. Shibahara, Y. Tomita, T. Sakakura, C. Nager, B. Chaudhuri, and R. Muller (1986) Nucl. Acids Res. 14:2413-2427). The DNA sequence encoded a protein predicted to be 58 kD after cleavage of a presumptive amino terminal signal sequence, in agreement with size estimates of purified mouse tyrosinase (J. B. Burnett (1971) Biol. Chem. 246:3079-3091). However, later studies disputed the authenticity of this clone. A second gene was cloned, that mapped to the c locus, and the authors claimed that the previous b locus gene was actually 5,6-hydroxyindole conversion factor, an accessory protein involved in mammalian melanogenesis (G. Muller, S. Ruppert, E. Schmid, and G. Schutz (1988) EMBO J. 7:2723-2730). The c locus gene encoded a protein of 58.5 kD, in general agreement with previous purified size estimates. Supporting these studies, the human tyrosinase gene was isolated from a cDNA library and also mapped to the c locus (B. Kwon, A. Haq, S. H. Pomerantz, and R. Halaban Proc. Natl. Acad. Sci., USA 84:7473-7477). Furthermore, individuals with oculocutaneous albinism have been shown to possess a missense mutation in the c locus (L. B. Giebel, K. M. Strunk, R. A. King, J. M. Hanifin, and R. A. Spritz (1990) Proc. Natl. Acad. Sci., USA 87:3255-3258).
The b locus and the c locus share a high degree of sequence conservation (86%) and also have the requisite copper-binding motifs of tyrosinases from Neurospora and Streptomyces (G. Muller, S. Ruppert, E. Schmid, and G. Schutz (1988) EMBO J. 7:2723-2730; S. Shibahara, Y. Tomita, T. Sakakura, C. Nager, B. Chaudhuri, and R. Muller (1986) Nucl. Acids Res. 14:2413-2427). More recent studies have further complicated the issue by revealing that both the b locus and the c locus gene products possess tyrosinase activity, and a locus encoding the TRP2 protein may be a third tyrosinase (M. Jimenez, K. Tsukamoto, and V. J. Hearing (1991) J. Biol. Chem. 266:1147-1156). These results suggest that a family of tyrosinases may mediate melanogenesis in mammals, each one functioning in a different aspect of the process. Reflecting this, the b locus protein is preferentially expressed in transformed melanocytes while the c locus is expressed at higher levels in normal melanocytes (M. Jimenez, K. Tsukamoto, and V. J. Hearing (1991) J. Biol. Chem. 266:1147-1156). The functions of the p, e, and A.sup.Y loci are yet to be determined but could be involved in regulation of tyrosinase(s).
The Streptomyces tyrosinase is the most extensively studied bacterial melanogenesis system. The ability to synthesize melanin is highly unstable and spontaneous mutants arise frequently (H. Schrempf (1983) Mol. Gen. Genet. 189:501-505). Two classes of mutants have been defined, Class I mutants defective in tyrosinase activity, and Class II mutants defective in tyrosine secretion (R. R. Crameri, L. Ettlinger, R. Hutter, K. Lerch, M. A. Suter, and J. A. Vetterli (1982) J. Gen. Microbiol. 128:371-379). Neither of these mutants demonstrated any detrimental effects from loss of melanin synthesis, suggesting that the gene was non-essential for growth in laboratory culture. The Class I mutants were further divided into three genetic loci mutable for tyrosinase gene expression (R. Crameri, G. Hintermann, R. Hutter, and T. Keiser (1984) Can. J. Microbiol. 30:1058-1067). The melA and melB loci were thought to be genes involved in the regulation of melanogenesis and melC the tyrosinase structural gene.
The tyrosinase genes for two species, S. glaucescens and S. antibioticus have been cloned and sequenced (V. Bernan, D. Filpula, W. Herber, M. Bibb and E. Katz (1985) Gene 37:101-110; G. Hintermann, M. Zatchez, and R. Hutter (1985) Mol. Gen. Genet. 200:422-432; M. Huber, G. Hintermann, and K. Lerch (1985) Biochemistry 24:6038-6044; E. Katz, C. J. Thompson, and D. A. Hopwood (1983) J. Gen. Micro. 129:2703-2714) and are the only bacterial melanogenesis genes reported to be cloned. The cloned fragments correspond to the melC locus defined in the earlier mutational analyses.
The role of melanin as an oxygen/free radical sink is an attractive hypothesis long proposed as a function for the polymer (A. A. Bell and M. H. Wheeler (1986) Ann. Rev. Phytopathol. 24:411-451). Most melanin synthesis systems, including those in Streptomyces, are dispensable for growth in the laboratory (H. Schrempf (1983) Mol. Gen. Genet. 189:501-505). Melanization may act as an oxygen/free radical sink, involved in coping with oxygen and oxidative stress. It is possible that other melanizing bacteria also benefit from the trapping of free radicals and/or the increased oxygen demand during melanin polymerization.
When S. colwelliana D is grown on marine agar plates, the melanin diffuses outward from the colonies. Production is greatly enhanced by the addition of tyrosine, suggesting that the pigment is derived from tyrosine metabolism (Fuqua et al, J. Gen. Microbiol. 139:1105-1114 (1993)).
Some Pseudomonas aeruginosa strains produce melanin-like pigments when growth on peptone agar. (Pigment Microbiology, P. X. Margalith, ed. Chapman & Hall, pp. 11-13 (1992)). This would not take place in tyrosine-free media. Since tyrosinase inhibitors (KCN, Na.sub.2 S) are without effect and DOPA can not be identified in culture extracts, there are doubts with regard to the identity of the pigment. All melanin-forming strains have been found to accumulate homogentisic acid (2,5-di-hydroxyphenylacetic acid), while melanin negative strains do not. It has been argued that the melanin strains were not tyrosinase-positive, but rather mutants defective in the metabolism of homogentisic acid. Under oxidative conditions in the presence of amino acids this was polymerized into a brown aeruginosa melanin. A similar brown pigment was shown to be produced by strains of Serratia marcescens when cultivated on tyrosine.
In this organism, melanogenesis is mediated primarily by MelA, the product of the melA gene. (Fuqua et al, Gene, 109:131-136 (1991); Fuqua et al, J. Gen. Microbiol. 139:1105-1114 (1993)). When E. coli express the cloned melA gene, they are transformed to a melanogenic phenotype, which is again enhanced by tyrosine in the medium (Fuqua et al, Gene 09:131-136 (1991); Fuqua et al, J. Gen. Microbiol., 139:1105-1114 (1993)). Analysis of lysates of S. colwelliana D and transformed E. coli demonstrate the accumulation of a dominant, electrochemically active intermediate, previously referred to as TyrP, (Fuqua, Doctoral dissertation. University of Maryland, College Park (1991)). a primary intermediate in pigment production by S. colwelliana D.
There are a variety of known pathways by which tyrosine can be converted to melanin. Melanins comprise a general class of complex, polyphenolic heteropolymers which are found as dark pigments in bacteria, fungi and higher organisms. Eumelanins are black and are synthesized by the classic Mason-Raper biosynthetic pathway in which a tyrosinase converts tyrosine to dihydroxyphenylalanine (DOPA) and then to dopaquinone which then autooxidizes and polymerizes to form eumelanin (Raper, Physiol Rev. 8:245-282 (1928)). Phaomelanins are brown, red or yellow pigments that form when dopaquinone reacts with glutathione or cysteine prior to further oxidation and polymerization. Structural analogues of tyrosine, or tyrosine metabolites, may also serve a s precursors for eumelanin and phaomelanin synthesis (Bell et al, Ann. Rev. Phytopathol. 24:411-451 (1986); Nicolaus, Melanins, Mermann, Paris (1968); Prota, Arch. Biochem. Biophys. 160:73-82 (1974)). Allomelanins, formed from catechol by a mechanism that is not well characterized, are described primarily from plants, but are also produced by bacteria. Pyomelanins, also called alkaptons, are produced from tyrosine through homogentisic acid (Yabuuchi et al, Int. J. Syst Bact. 22:53-65 (1972)). While most melanins fall into these categories, other types of melanins exist. (Bell et al, Ann. Rev. Phytopathol. 24:411-451 (1986)).
Bacteria are known to produce phaomelanins (Ivins et al, Infect. Immun. 34:895-899 (1981)) and pyomelanins (Yabuuchi et al, Int. J. Syst Bact. 22:53-65 (1972)). Melanin synthesis in bacteria has been most intensively studied in Streptomyces spp. (Lerch et al, Eur. J. Biochem. 52:125-138 (1981); Lerch et al, Eur. J. Biochem. 31:427-437 (1972)). These, and other bacteria (Aurstad et al, Acta Vet. Scand. 13:251-259 (1972); Pomerantz et al, Arch. Biochem. Biophys. 160:73-82 (1988)), synthesize a eumelanin via the action of tyrosinase on tyrosine via the MasonRaper pathway.
Preliminary experiments into the melanogenic pathway of S. colwelliana D indicated that TyrP was not an intermediate in the Mason-Raper melanogenic pathway. For example, TyrP was detected neither as a product of mushroom tyrosinase action on tyrosine, nor when DOPA was oxidized under a variety of conditions. TyrP was not found to coelute with authentic standards of known Mason-Raper intermediates or a multitude of related compounds.